It is known that it is desirable to direct expression of a gene of interest ("GOI") in certain tissues of an organism--such as a filamentous fungus (such as Aspergillus niger) or even a plant crop. The resultant protein or enzyme may be useful for the organism itself. For example, it may be desirable to produce crop protein products with an optimised amino acid composition and so increase the nutritive value of a crop. For example, the crop may be made more useful as a feed. In the alternative, it may be desirable to isolate the resultant protein or enzyme and then use the protein or enzyme to prepare, for example, food compositions. In this regard, the resultant protein or enzyme can be a component of the food composition or it can be used to prepare food compositions, including altering the characteristics or appearance of food compositions.
It may even be desirable to use the organism, such as a filamentous fungus or a crop plant, to express non-plant genes, such as for the same purposes.
Also, it may be desirable to use an organism, such as a filamentous fungus or a crop plant, to express mammalian genes. Examples of the latter products include interferons, insulin, blood factors and plasminogen activators.
It is also desirable to use micro-organisms, such as filamentous fungi, to prepare products from GOIs by use of promoters that are active in the micro-organisms.
Fruit and vegetable cell walls largely consist of polysaccharide, the major components being pectin, cellulose and xyloglucan, R. R. Selvendran and J. A. Robertson, IFR Report 1989. Numerous cell wall models have been proposed which attempt to incorporate the essential properties of strength and flexibility (P. Albersheim, Sci. Am. 232, 81-95, 1975;, P. Albersheim, Plant Biochem. 3rd Edition (Bonner and Varner), Ac. Press, 1976; T. Hayashi, Ann. Rev. Plant Physiol. & Plant Mol. Biol., 40, 139-168, 1989).
The composition of the plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various .beta.-xylan chains, such as xyloglucans) and pectic substances (consisting of galacturonans and rhamnogalacturonans; arabinans; and galactans and arabinogalactans).
In particular, glucans are polysaccharides made up exclusively of glucose subunits. Typical examples of glucans are starch and cellulose.
The enzymes that degrade glucans are collectively referred to as glucanases. A typical glucanase is .beta.-1,4-endoglucanase.
.beta.-1,4-endoglucanases have uses in many industries. For example, in the brewing industry, barley is used for production of malt, and, in the latter years, as adjunct in the brewing process. When the quality of the malt is poor, or barley has been used as an adjunct, problems with high viscosity in the wort can arise because of .beta.-glucans from the barley. In this regard, barley contains large quantities of mixed .beta.-1,3/1,4-glucans of very high molecular weight. When dissolved, these glucans produce high viscosity solutions, which can cause troubles in some applications. For example, the high viscosity reduces the filterability of the wort and can lead to unacceptable long filtration times. To avoid these problems .beta.-glucanase has been traditionally added to wort to avoid such problems--i.e. the problem with glucans can be avoided by addition of enzymes, in particular, glucanases, which degrade the polymers.
Further information on these problems may be found in the Grindsted brochure called "Glucanase GV", the reviews by Dr. C. W. Bamforth (Brewers Digest June 1982 pages 22-28; and Brewers' Guardian September 1985 pages 21-26), and the paper by T. Godfrey (Industrial Enzymology The Application of Enzymes in Industry Chapter 4.5 pages 221-259).
In the feed industry barley can be used for chicken feed because it is cheap, but again the .beta.-glucan can give problems for the digestion of the chicken. By addition of .beta.-glucanase to the feed the digestibility of the feed can be increased. In addition, the faeces of chickens feeding on feed containing barley is sticky making it difficult to remove and results in dirty eggs.
WO 93/2019 discusses endo-.beta.-1,4-glucanases (EC no. 3.2.1.4). According to WO 93/2019, these glucanases are a group of hydrolases which catalyse endo hydrolysis of 1,4-.beta.-D-glycosidic linkages in cellulose, lichenin, cereal .beta.-D-glucans and other plant material containing cellulosic parts. Endo-1,4-.beta.-D-glucan 4-glucano hydrolase is sometimes called endo-.beta.-1,4-glucanase.
The endo-.beta.-1,4-glucanase of WO 93/2019 exhibits a pH-optimum of 2.0 to 4.0, an isoelectric point of 2.0 to 3.5, a molecular weight of between 30,000 and 50,000, and a temperature optimum between 30 and 70.degree. C.
Further teachings on glucans may be found in WO 93/17101, in particular xyloglucans. According to WO 93/17101 the xyloglucans are 1,4-.beta.-glucans that have been extensively substituted with .alpha.-1,6-xylosyl side chains, some of which are 1,2-.beta.-galactosylated. They are found in large amounts in the primary cell walls of dicots but also in certain seeds, where they serve different roles. Primary cell wall xyloglucan is fucosylated. Xyloglucan is tightly hydrogen bonded to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it. Xyloglucan is thought to form cross-bridoes between cellulose microfibrils, the cellulose/xyloglucan network forming the major load-bearing/elastic network of the wall. DCB mutated suspension culture cells (cell walls lacking cellulose) release xyloglucan into their media, suggesting that xyloglucan is normally rightly bound to cellulose.
Hydrolysis of primary cell wall xyloglucan has been demonstrated in segments of dark grown squash hypocotyls, during IAA induced growth (K. Wakabayashi et al, Plant Physiol., 95, 1070-1076, 1991). Endohydrolysis of wall xyloglucan is thought to contribute to wall loosening which accompanies cell expansion (T. Hyashi, Ann. Rev. Plant Physiol. & Plant Mol. Biol., 40, 139-168, 1989). The average molecular weight of xyloglucan has also been shown to decrease during tomato fruit ripening and this may contribute to the tissue softening which accompanies the ripening process (D. J. Huber, J. Amer. Soc. Hort. Sci., 108(3), 405-409, 1983). Certain seeds, e.g. Nasturtium, contain up to 30% by weight of xyloglucan, stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination.
It would be useful to increase glucanase activity, for example to have a plant with high concentration of glucanase for use in feed, preferably using recombinant DNA techniques.